The human homologue of Notch1 was originally identified at the chromosomal breakpoint in a recurrent chromosomal translocation in T-cell leukemia. We have previously shown that dysregulated expression of Notch1 causes T-cell leukemia in mice and that its role in leukemia may be related to its normal function in hematopoiesis. As part of our long term plan to understand the role of Notch in leukemia, we have undertaken studies to identify Notch function in hematopoiesis. Notch proteins are a conserved family that regulates cell fate choice in many lineages, including stem cells. Recently, we and others have provided evidence that Notch plays a key role in regulating B/T cell fate decisions and later stages of T cell development. The proposed studies focus on how Notch proteins influence B/T lineage determination and later stages of T and B cell development. In Specific Aim 1, retroviral transduction of BM stem cells with Notch1 mutants or a putative downstream effector of Notch activity will be used to investigate the signaling pathways responsible for B/T cell fate decisions. In Specific Aim 2, transduction of stem cells and thymocyte subpopulations from wild type and knockout mice in conjunction with radiation chimeras, fetal thymic organ cultures, intrathymic injections, and proliferation and apoptosis assays will be used to determine the propensity of Notch1 to modulate early T cell development, the mechanism by which activated Notch1 influences CD4+CD8+ homeostasis, and how Notch1 activity regulates CD4+CD8+ cell maturation. In Specific Aim 3, transduction of pro-B cells and later stage B-lineage cells with Notch1-4 and Notch1 mutants will be used to investigate the mechanism by which Notch l inhibits B cell development and to determine whether other Notch family members play a similar or unique role in B lymphopoiesis. These studies will lead to an improved understanding of of normal hematopoiesis, and in doing so, will provide therapeutic insights into treating leukemia and other diseases of the immune and hematopoietic systems.